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IP06 Protein A Agarose

Protein A Agarose MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
IP06
  
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Overview

Replacement Information
Description
Overview

This product has been discontinued.





Designed for immunoglobulin purification at low pressure. Product size refers to volume of packed beads.
Catalogue NumberIP06
Brand Family Calbiochem®
References
Product Information
FormLiquid slurry
Formulation50% suspension in PBS.
Preservative≤0.1% sodium azide
Applications
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Application NotesAntibody Purification
Application CommentsNote: the product size refers to the volume of packed beads. This product can be used directly with serum, plasma, tissue culture media, ascites, or other biological fluids, but if sufficient quantities of starting material are available we recommend an initial clean up step. The column life will be greatly extended if aggregated proteins and lipids are removed from the immunoglobulin in the clean up step. Use 5-10 ml of packed beads per ml serum.

Recommended Protocol for IgG Purification

Buffers

All concentrations stated are for working solutions, not the 10X concentrates. Caution: sodium azide is poison.

• Binding/Washing Buffer: 100 mM sodium phosphate pH 7.0, 150 mM sodium chloride, 5 mM sodium EDTA, 0.01% sodium azide.
• Elution Buffer A (see Note section): 500 mM ammonium acetate pH 3.0, 0.01% sodium azide.
• Elution Buffer B: 10 mM glycine/HCl pH 3.0, and 0.01% sodium azide.
• Neutralization Buffer : 500 mM Tris Base, 0.01% sodium azide.
• Storage Buffer: 100 mM sodium phosphate, pH 7.0, 0.01% sodium azide.

Protocol

A. Clean Up and Concentration
Ascites and serum should be clotted at room temperature, refrigerated at 4°C overnight (to allow the clot to shrink and lipids to separate), and centrifuged multiple times to remove all clotted protein and lipid. Remove the lipid from the top of the centrifuge tube with a glass rod or small wooden stick. Tissue culture media should be centrifuged or filtered to remove aggregates.
IgG can be concentrated and partially purified by use of an ammonium sulfate precipitation step. Add ammonium sulfate to 50% saturation (313 g per L) with stirring and check the pH adjusting to 7.0 by addition of 1 M HCl or NaOH. Centrifuge to collect precipitated immunoglobulin, dissolve in binding buffer and dialyze against the same buffer.

B. Purification
1. Pack a column with the Agarose Conjugate.
2. Wash with about 20 column volumes Washing/Binding Buffer until pH of eluate is 7.0.
3. If IgG has not been previously dialyzed against binding buffers dilute or dialyze IgG-containing sample into the Washing/Binding Buffer (pH 6.5-7.5).
4. Load sample onto column.
5. Wash with Washing/Binding Buffer until the absorbance of the eluate at 280 nm approaches background level.
6. Wash with Elution Buffer A to elute IgG, and collect fractions until A280 returns to background levels.
7. Wash with Elution Buffer B, and collect fractions until A280 returns to background. Most IgG should elute with buffer A.
8. Neutralize eluted IgG fractions by addition of an equal volume of neutralization buffer and check the pH with pH paper. For best results, neutralize eluate promptly.
9. To re-use the column immediately, repeat procedure from Step 2.
10. To prepare the column for storage, wash column with 5 column volumes of Elution Buffer B.
11. To store column wash with 30 column volumes storage buffer; then seal column outlets and store in refrigerator.
12. Quantitate the purified IgG using the formula:

Absorbance at 280 nm/1.4 = Concentration (mg/ml).

Note: To make Elution Buffer A, start with acetic acid and adjust the pH to 3.0 with ammonium hydroxide.
Biological Information
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code Blue Ice Only
Toxicity Standard Handling
Storage +2°C to +8°C
Do not freeze Yes
Packaging Information
Transport Information
Supplemental Information
Specifications
Global Trade Item Number
Catalogue Number GTIN
IP06 0

Documentation

Protein A Agarose Certificates of Analysis

TitleLot Number
IP06
Data Sheet

Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

Revision10-June-2010 RFH
ApplicationAntibody Purification
DescriptionProtein A covalently conjugated to agarose. Useful for the purification of IgG from biological fluids.
BackgroundThe protein is covalently coupled to the agarose support and does not leach. The protein A will bind IgG from biological fluids, but with different efficiencies, depending on the species. In general, Protein A works well for purification of IgG of large mammals, while Protein G PLUS and a mixture of Protein G PLUS with Protein A are best for rodent IgG purifications.
FormLiquid slurry
Formulation50% suspension in PBS.
Preservative≤0.1% sodium azide
CommentsNote: the product size refers to the volume of packed beads. This product can be used directly with serum, plasma, tissue culture media, ascites, or other biological fluids, but if sufficient quantities of starting material are available we recommend an initial clean up step. The column life will be greatly extended if aggregated proteins and lipids are removed from the immunoglobulin in the clean up step. Use 5-10 ml of packed beads per ml serum.

Recommended Protocol for IgG Purification

Buffers

All concentrations stated are for working solutions, not the 10X concentrates. Caution: sodium azide is poison.

• Binding/Washing Buffer: 100 mM sodium phosphate pH 7.0, 150 mM sodium chloride, 5 mM sodium EDTA, 0.01% sodium azide.
• Elution Buffer A (see Note section): 500 mM ammonium acetate pH 3.0, 0.01% sodium azide.
• Elution Buffer B: 10 mM glycine/HCl pH 3.0, and 0.01% sodium azide.
• Neutralization Buffer : 500 mM Tris Base, 0.01% sodium azide.
• Storage Buffer: 100 mM sodium phosphate, pH 7.0, 0.01% sodium azide.

Protocol

A. Clean Up and Concentration
Ascites and serum should be clotted at room temperature, refrigerated at 4°C overnight (to allow the clot to shrink and lipids to separate), and centrifuged multiple times to remove all clotted protein and lipid. Remove the lipid from the top of the centrifuge tube with a glass rod or small wooden stick. Tissue culture media should be centrifuged or filtered to remove aggregates.
IgG can be concentrated and partially purified by use of an ammonium sulfate precipitation step. Add ammonium sulfate to 50% saturation (313 g per L) with stirring and check the pH adjusting to 7.0 by addition of 1 M HCl or NaOH. Centrifuge to collect precipitated immunoglobulin, dissolve in binding buffer and dialyze against the same buffer.

B. Purification
1. Pack a column with the Agarose Conjugate.
2. Wash with about 20 column volumes Washing/Binding Buffer until pH of eluate is 7.0.
3. If IgG has not been previously dialyzed against binding buffers dilute or dialyze IgG-containing sample into the Washing/Binding Buffer (pH 6.5-7.5).
4. Load sample onto column.
5. Wash with Washing/Binding Buffer until the absorbance of the eluate at 280 nm approaches background level.
6. Wash with Elution Buffer A to elute IgG, and collect fractions until A280 returns to background levels.
7. Wash with Elution Buffer B, and collect fractions until A280 returns to background. Most IgG should elute with buffer A.
8. Neutralize eluted IgG fractions by addition of an equal volume of neutralization buffer and check the pH with pH paper. For best results, neutralize eluate promptly.
9. To re-use the column immediately, repeat procedure from Step 2.
10. To prepare the column for storage, wash column with 5 column volumes of Elution Buffer B.
11. To store column wash with 30 column volumes storage buffer; then seal column outlets and store in refrigerator.
12. Quantitate the purified IgG using the formula:

Absorbance at 280 nm/1.4 = Concentration (mg/ml).

Note: To make Elution Buffer A, start with acetic acid and adjust the pH to 3.0 with ammonium hydroxide.
Storage +2°C to +8°C
Do Not Freeze Yes
Toxicity Standard Handling

Related Products & Applications

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Related Products

Catalogue Number Description
IP02 Protein A Agarose Suspension
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IP10 Protein G Plus/Protein A-Agarose

Categories

Life Science Research > Protein Sample Preparation > Protein Purification > Agarose Bead Affinity Purification > Agarose Beads for IP & Antibody Purification
Life Science Research > Protein Detection and Quantification > Immunoassays > Immunoprecipitation (IP) > Agarose Beads for IP & Antibody Purification
Life Science Research > Antibodies and Assays > Immunoassays > Immunoprecipitation (IP) > Agarose Beads for IP & Antibody Purification